The original objectives of the proposal were: 1. Compare mutants 444 and 710 to Rev1 (parent strain), and 16M (field strain) in murine and human macrophage lines for phenotypic differences. 2. Determine in vivo virulence and survival of the mutants 444 and 710 in guinea pigs and mice. 3. Determine humoral and cell-mediated immune responses induced by mutants 444 and 710 in guinea pigs and mice. 4. Determine in vivo protection of mice and guinea pigs provided by mutants 444 and 710 compared to Rev1. Background: While human and animal brucellosis are rare in the U.S., brucellosis caused by B. melitensis remains relatively constant in Israel. Despite a national campaign to control brucellosis in Israel, the misuse of Rev1 Elberg vaccine strain among pregnant animals has produced abortion storms raising concern of human infection due to vaccine excretion in the milk. Further, some commercial Rev1 vaccine lots can: a) produce persistent infection, b) infect humans, c) be horizontally transmitted, d) cause abortion, and e) induce a persistent anti-O-polysaccharide antibody response confounding the distinction between infected and vaccinated animals. In Israel, vaccination practices have not optimally protected the milk supply from Brucella and Rev 1 vaccine can exacerbate the problem. In addition, cattle vaccinated against B. abortus are not protected against B. melitensis supporting the need for an improved vaccine. A safe vaccine used in adult animals to produce herd resistance to infection and a vaccine that can be distinguished from virulent infection is needed. A rough Rev1 vaccine would be less virulent than the parental smooth strain and permit serologic distinction between vaccinated and infected animals. Advantages of the Rev1 vaccine foundation are: 1) Rev1 vaccination of sheep and goats against B. melintensisis approved; therefore, vaccines derived from the Rev1 foundation may be readily accepted by licensing agencies as well as commercial companies, and 2) considerable data exists on Rev1vaccination and Rev1 proteins. Therefore, a post-genomic vaccine against B. melitensis based on the Rev1 foundation would provide a great advantage. Major conclusions from our work are: 1) We have determined that mutant 710 is highly attenuated in macrophages compared to virulent field strain 16M and mutant 444. 2) We have confirmed that mutant 710 is highly attenuated in guinea pigs and mice. 3) We have determined immune responses induced by mutant 710 in animals. 4) We have determined in vivo protection of mice and guinea pigs provided by mutants 444 and 710 compared to Rev1, and importantly, mutant 710 provides a high level of protection against challenge with virulent B. melitensis 16M. Thus, our data support the goals of the grant and provide the foundation for a future vaccine useful against B. melitensis in Israel. Because of patent considerations, many of our findings with 444 and 710 have not yet been published. Scientific and Agricultural Implications: Our findings support the development of a vaccine against B. melitensis based on the mutant 710. Because strain 710 is a mutant of the Elberg Rev1 vaccine, commercialization is more likely than development of an entirely new, uncharacterized Brucella mutant or strain.